''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]]. Please contribute.''

[http://www.openwetware.org/wiki/User:Korey_Griffin Korey Griffin]

[http://www.openwetware.org/wiki/User:Korey_Griffin Korey Griffin]

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Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;

Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;

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==Reagents I==

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[[Image:nucleusmodel.jpg]]

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The nucleus of a eukaryotic cell

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==Hattori et al nuclear extract preparation==

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===Reagents I===

'''I. Homogenization buffer:'''

'''I. Homogenization buffer:'''

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10ml of ethanol

10ml of ethanol

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==Procedure I==

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===Procedure I===

'''I. Homogenize cells or tissue'''

'''I. Homogenize cells or tissue'''

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3. Measure protein and aliquot supernatant (100-500mg/tube) and store it at -70C

3. Measure protein and aliquot supernatant (100-500mg/tube) and store it at -70C

Procedure I

I. Homogenize cells or tissue
1. Wash commercially available HeLa cells (30g) or minced in 3mm pieces fresh liver(15-20g) from staining with homogenizing buffer (10ml) and transfer it to 100ml beaker
2. Add to the beaker another 25ml of the buffer and split the content in half (2x15ml)
3. Homogenize each portion twice, 10 strokes (“up” and “down”) each with a teflon pestle. Between strokes, cool homogenizer on ice. Try to maintain temperature close to +4C

II. Pellet the nuclei
1. Filter homogenate through the four- layer cloth (25-30ml) and mix homogenate with cushion buffer (50ml of cushion buffer per 25ml of the homogenate
2. Distribute to ultracentrifuge tubes cushion buffer (10 ml of buffer per a tube). Carefully load cushion buffer- homogenate mix on the top (total volume is about 38 ml).
3 Equilibrate tubes at the balance and centrifuge them in SW28 rotor (24,000 rpm or 76220g, 50min, 1C) or 60Ti rotor (same time and temperature at 32,750 rpm/min)

Procedure II

Hela cells were grown in spinner flasks at 37oC in Joklik’s MEM containing 5% calf serum. They were frown to 4 to 6 x 105 cells per ml prior to harvesting for extract preparation.

HeLa cells were harvested from cell culture media by centrifugation (at room temperature) for 10 min at 2000 rpm in a Sorvall HG4L rotor. Pelleted cells were then suspended in five volumes of 4oC phosphate buffered saline and collected by centrifugation as detailed above; subsequent steps were performed at 4oC.

The cells were suspended in five packed cell pellet volumes of buffer A and allowed to stand for 10 min.

The cells were collected by centrifugation as before and suspended in two packed cell pellet volumes (volume prior to the initial wash with buffer A) of buffer A and lysed by 10 strokes of a Kontes all glass Dounce homogenizer (B type pestle).

The homogenate was checked microscopically for cell lysis and centrifuged for 10 min at 2000 rpm in a Sorvall HG4L rotor to pellet nuclei.

The supernatant was carefully decanted, mixed with 0.11 volumes of buffer B, and centrifuged for 60 min at 100,000 gav (Beckman Type 42 rotor). The high speed supernatant from this step was dialyzed five to eight hours against 20 volumes of buffer D and is designated the S100 fraction.

The nuclear extract was prepared as follows. The pellet obtained from the low speed centrifugation of the homogenate was subjected to a second centrifugation for 20 min at 25,000 gav (Sorvall SS34 rotor), to remove residual cytoplasmic material and this pellet was designated as crude nuclei.

These crude nuclei were resuspended in 3 ml of buffer C per 109 cells with a Kontes all glass Dounce homogenizer (10 strokes with a type B pestle). The resulting suspension was stirred gently with a magnetic stirring bar for 30 min and then centrifuged for 30 min at 25,000 gav (Sorvall SS34 rotor).

The resulting clear supernatant was dialyzed against 50 volumes of buffer D for five hours. The dialysate was centrifuged at 25,000 gav (Sorvall SS34 rotor) for 20 min and the resulting precipitate discarded.

The supernatant, designated the nuclear extract, was frozen as aliquots in liquid nitrogen and stored at -80oC.

The protein concentration was usually 6-8 mg/ml and 15-20 mg of protein were obtained from 109 cells.